Label‐free quantitative proteome analysis of skeletal tissues under mechanical load

Skeletal tissue has the capability to adapt its mass and structure in response to mechanical stress. However, the molecular mechanism of bone and cartilage to respond to mechanical stress are not fully understood. A label‐free quantitative proteome approach was used for the first time to obtain a global perspective of the response of skeletal tissue to mechanical stress. Label‐free quantitative analysis of 1D‐PAGE‐LC/MS/MS based proteomics was applied to identify differentially expressed proteins. Differential expression analysis in the experimental groups and control group showed significant changes for 248 proteins including proteins related to proliferation, differentiation, regulation of signal transduction and energy metabolic pathways. Fluorescence labeling by incorporation of alizarin/calcein in newly formed bone minerals qualitatively demonstrated new bone formation. Skeletal tissues under mechanical load evoked marked new bone formation in comparison with the control group. Bone material apposition was evident. Our data suggest that 39 proteins were assigned a role in anabolic process. Comparisons of anabolic versus catabolic features of the proteomes show that 42 proteins were related to catabolic. In addition, some proteins were related to regulation of signal transduction and energy pathways, such as tropomyosin 4, fibronectin 1, and laminin, might be new molecular targets that are responsive to mechanical force. Differentially expressed proteins identified in this model may offer a useful starting point for elucidating novel aspects of the effects of mechanical force on skeletal tissue. J. Cell. Biochem. 108: 600–611, 2009. © 2009 Wiley‐Liss, Inc.     

Advanced monitoring of high-rate anaerobic reactors through quantitative image analysis of granular sludge and multivariate statistical analysis

Four organic loading disturbances were performed in lab-scale EGSB reactors fed with ethanol. In load disturbance 1 (LD1) and 2 (LD2), the organic loading rate (OLR) was increased between 5 and 18.5 kg COD m(-3) day(-1), through the influent ethanol concentration increase, and the hydraulic retention time decrease from 7.8 to 2.5 h, respectively. Load disturbances 3 (LD3) and 4 (LD4) were applied by increasing the OLR to 50 kg COD m(-3) day(-1) during 3 days and 16 days, respectively. The granular sludge morphology was quantified by image analysis and was related to the reactor performance, including effluent volatile suspended solids, indicator of washout events. In general, it was observed the selective washout of filamentous forms associated to granules erosion/fragmentation and to a decrease in the specific acetoclastic activity. These phenomena induced the transitory deterioration of reactor performance in LD2, LD3, and LD4, but not in LD1. Extending the exposure time in LD4 promoted acetogenesis inhibition after 144 h. The application of Principal Components Analysis determined a latent variable that encompasses a weighted sum of performance, physiological and morphological information. This new variable was highly sensitive to reactor efficiency deterioration, enclosing variations between 27% and 268% in the first hours of disturbances. The high loadings raised by image analysis parameters, especially filaments length per aggregates area (LfA), revealed that morphological changes of granular sludge, should be considered to monitor and control load disturbances in high rate anaerobic (granular) sludge bed digesters.     

Mechanical regulation of HB-GAM expression in bone cells

Bone adaption upon mechanical stimulation is accompanied by changes in gene expression. In this context we investigated the influence of mechanical loading on heparin binding growth associated molecule (HB-GAM) expression, an extracellular matrix molecule which in cell culture has been shown to stimulate the differentiation of osteoblasts. We obtained information on the participating signal transduction pathways using a mitogenic loading regimen. Specific inhibitors of various signal transduction pathways were added to loaded cells and to unloaded controls. By semi-quantitative PCR studies we demonstrated a rapid decrease of HB-GAM expression in primary osteoblasts and SaOs-2 cells by 20-30% upon mechanical loading within 30min. We showed that the RGD-integrin interaction is involved in the regulation of HB-GAM expression. Furthermore, integrity of the cytoskeleton, stretch-activated, and voltage-sensitive Ca(2+) channels as well as gap junctional communication are necessary for the downregulation of HB-GAM expression by mechanical loading.     

Denitrification and nitrous oxide emissions from riparian forests soils exposed to prolonged nitrogen runoff

Compared to upland forests, riparian forest soils have greater potential to remove nitrate (NO₃) from agricultural runoff through denitrification. It is unclear, however, whether prolonged exposure of riparian soils to nitrogen (N) loading will affect the rate of denitrification and its end products. This research assesses the rate of denitrification and nitrous oxide (N₂O) emissions from riparian forest soils exposed to prolonged nutrient runoff from plant nurseries and compares these to similar forest soils not exposed to nutrient runoff. Nursery runoff also contains high levels of phosphate (PO₄). Since there are conflicting reports on the impact of PO₄ on the activity of denitrifying microbes, the impact of PO₄ on such activity was also investigated. Bulk and intact soil cores were collected from N-exposed and non-exposed forests to determine denitrification and N₂O emission rates, whereas denitrification potential was determined using soil slurries. Compared to the non-amended treatment, denitrification rate increased 2.7- and 3.4-fold when soil cores collected from both N-exposed and non-exposed sites were amended with 30 and 60 μg NO₃-N g⁻¹ soil, respectively. Net N₂O emissions were 1.5 and 1.7 times higher from the N-exposed sites compared to the non-exposed sites at 30 and 60 μg NO₃-N g⁻¹ soil amendment rates, respectively. Similarly, denitrification potential increased 17 times in response to addition of 15 μg NO₃-N g⁻¹ in soil slurries. The addition of PO₄ (5 μg PO₄-P g⁻¹) to soil slurries and intact cores did not affect denitrification rates. These observations suggest that prolonged N loading did not affect the denitrification potential of the riparian forest soils; however, it did result in higher N₂O emissions compared to emission rates from non-exposed forest soils.     

Renal function and plasma arginine vasotocin during an acute salt load in feral chickens

Summary Renal clearance experiments were conducted on feral chickens descended from birds collected from a coral island off the coast of Queensland, Australia. Following a control period when 0.13 M NaCl was used as a vehicle for the renal function markers, a salt load was imposed by infusion of 1 M NaCl. The hypertonic NaCl infusion resulted in increases in glomerular filtration rate (GFR), effective renal blood flow (ERBF), and urine flow rate (V), whereas filtration fraction decreased. Haematocrit was reduced and plasma osmolality, sodium, chloride and potassium concentrations increased. Plasma arginine vasotocin (P_AVT) levels increased from 31.4±2.3 pg·ml^-1 during the control infusion to 56.0±1.7 pg·ml^-1 following a salt load of 12 mmol Nacl·kg^-1 The sensitivity of release of AVT was 0.69±0.11 pg·ml^-1 per mosmol·kg^-1. The concentrations of Na and Cl in urine increased, whereas urine osmolality and potassium concentration decreased. The expansion of the extracellular fluid volume induced by the salt loading would tend to suppress the release of AVT, whereas the osmotic stimulus would provide a stimulus for the release of AVT. In this study, GFR, ERBF and ERPF increased at the same time as P_AVT increased.Abbreviations AVP arginine vasopressin - AVT arginine vasotocin - ERBF effective renal blood flow - ERPF effective renal plasma flow - GFR glomerular filtration rate - P_avt plasma arginine vasotocin concentration - PAH paraaminohippuric acid - SEM standard error of mean - SNGER single nephron glomerular filtration rate - U/P urine to plasma ratio - V urine flow rate     

Trade‐off between flight capability and reproduction in male Velarifictorus asperses crickets

1. There are numerous data that support the trade‐off between flight capability and reproduction in female wing polymorphic insects, but the relationship between wing form and fitness remains poorly investigated in males. 2. In the present study, the development of flight muscle and gonads, spermatophore size, and multiple copulation ability were investigated in both long‐winged (LW) and short‐winged (SW) males to verify this trade‐off, using a wing dimorphic cricket species Velarifictorus aspersus (Walker). 3. The LW males had better‐developed wing muscles than the SW males on the day of emergence, and both of them developed wing muscles after emergence, but the peak of weight in SW males was achieved 4 days later than that of the LW males. The accessory glands (AG) of the LW males developed significantly slower than that of the SW males. These results suggest that development and maintenance of flight muscles have a cost on the development of reproductive organs in male V. asperses. 4. The SW males produced significantly heavier spermatophores in a single copulation and mated more often than LW males. This indicates the SW males have a higher mating success than the LW males, thereby increasing their chance of siring offspring.     

Coordination of wing motion and walking suggests common control of zigzag motor program in a male silkworm moth

The male silkworm moth, Bombyx mori, exhibits a zigzagging pattern as it walks upwind to pheromones. This species usually does not fly, but obvious wing-beating accompanies the pheromone-mediated walking. Males supported by a `sled', after having their legs removed, also moved upwind in a pheromone plume along zigzagging tracks, indicating that wing-generated thrust and torque result in locomotory paths similar to those observed from walking moths. Using a high-speed video system we investigated the correlation between the wing movements and zigzag walking. The wing ipsilateral to the direction of the turn showed a greater degree of retraction with respect to the contralateral wing. The timing of the wing retraction pattern was synchronized with changes of direction in the walking track. Coordination of wing movements and walking pattern was not dependent on visual feedback or sensory feedback generated from neck movements associated with turning. The results presented here, taken together with our previous studies of descending interneurons suggest that the coordination of wing movements with the walking pattern may result from the activity of a set of identified interneurons descending from the brain to the thoracic ganglia and/or may be coordinated by coupling of oscillating circuits for walking and wing beating. Accepted: 15 May 1997     

Improvement of pea biomass and seed productivity by simultaneous increase of phloem and embryo loading with amino acids

The development of sink organs such as fruits and seeds strongly depends on the amount of nitrogen that is moved within the phloem from photosynthetic‐active source leaves to the reproductive sinks. In many plant species nitrogen is transported as amino acids. In pea (Pisum sativum L.), source to sink partitioning of amino acids requires at least two active transport events mediated by plasma membrane‐localized proteins, and these are: (i) amino acid phloem loading; and (ii) import of amino acids into the seed cotyledons via epidermal transfer cells. As each of these transport steps might potentially be limiting to efficient nitrogen delivery to the pea embryo, we manipulated both simultaneously. Additional copies of the pea amino acid permease PsAAP1 were introduced into the pea genome and expression of the transporter was targeted to the sieve element‐companion cell complexes of the leaf phloem and to the epidermis of the seed cotyledons. The transgenic pea plants showed increased phloem loading and embryo loading of amino acids resulting in improved long distance transport of nitrogen, sink development and seed protein accumulation. Analyses of root and leaf tissues further revealed that genetic manipulation positively affected root nitrogen uptake, as well as primary source and sink metabolism. Overall, the results suggest that amino acid phloem loading exerts regulatory control over pea biomass production and seed yield, and that import of amino acids into the cotyledons limits seed protein levels.     

Application of a hybrid biofilter column for the removal of Cr(VI) from aqueous solution using an indigenous bacterial strain Pseudomonas taiwanensis

In the present study, a laboratory-scale biofilter column was designed and fabricated. It was packed with a mixture of coal and compost as a packing medium. The column was enriched with an indigenous bacterial strain Pseudomonas taiwanensis isolated from aerobic mixed culture of Sewage Treatment Plant, BITS-Pilani, Pilani campus. The removal of hexavalent chromium [Cr(VI)] from aqueous solution was investigated in the biofilter column. The entire biofiltration operation was divided into five phases (I to V) for a period of 63 days. Biofilter column was subjected to shock loading conditions for 20 days immediately after 63 days of operation. The maximum removal efficiency of 89.4% was obtained during phase V for Cr(VI) inlet concentration of 40 mg L^?1. During shock loading, maximum removal efficiency was obtained as 90% for 48.5–50 mg L^?1 of initial Cr(VI) concentration. Kinetic parameters of biofiltration process for Cr(VI) removal were also determined by fitting Michaelis-Menten kinetic model with experimental data. The Michaelis-Menten kinetic constants were obtained as 0.258 mg L^?1 min^?1 and 26.83 mg L^?1. It was found that Ottengraf-Van den Oever model with zero-order diffusion limitation fit the experimental data quite well for phases III, IV, and V with coefficient of determination (R²) values .97, .99, and .984, respectively. A possible method for safe disposal of packing medium was also presented in this study.     

Loading and translocation of assimilate in the fine veins of sunflower leaves

Abstract The time course of loading and transport of assimilate in sunflower leaves was examined by pulse labelling with ¹⁴CO₂, followed by freeze drying or freeze substitution, and dry autoradiography at both low and high resolution. The five classes of veins, V1-V5 (V5 being smallest), show a division of function: V5 and V4 are engaged in loading and short distance transport; V3 to V1, in long distance translocation. The first high concentration of ¹⁴C is found in two or three phloem parenchyma cells (intermediary cells) of V5 and V4 veins. The sieve elements of V5 and V4 veins do not show comparable concentrations of ¹⁴C at any time. Recently assimilated ¹⁴C is transported by the intermediary cells for distances of about 0.5 mm to the V3 veins. In V3 to V1 veins translocation is in the sieve tubes. Transport in V5 and V4 veins is in two directions, that in V3 to V1, in one direction towards the petiole. The high concentration of ¹⁴C formed in the intermediary cells does not increase further as the assimilate moves to the sieve tubes of the V3 veins, and so is probably the origin of the gradient that drives translocation.